The broad, long-term objective of the PI is to achieve ultrasensitivity in amperometric biosensing. This project is the first step toward this direction. The specific aims of this project are: 1. To assemble protein/enzyme molecules directly on the bare surface of nanoscale spatially confined regions on electrodes to reduce denaturation of the molecules and to form nanoscale (from 1 mu m to 100 nm) assemblies, 2. To demonstrate reduced protein denaturation and enhanced (several orders of magnitude) electron transfer across the molecule-electrode interface of the nanoscale assemblies. To accomplish specific aim 1, SPM-based fabrication techniques will be used to generate nanoscale templates on the surface of semiconductor and metallic electrodes. The templates will contain nanoscale spatially confined regions. Protein/enzyrnes molecules will be immobilized using different mechanisms into these regions to make physical contact with the bare surface of the electrodes. In-situ SPM imaging with submolecular resolution will be used to characterize the immobilized molecules in relation to preservation of their native conformation.To accomplish specific aim 2, electrochemical characterization of the nanoscale assemblies will be carried out to measure the current flowing across the molecule-electrode interface. Electrochemical characterization will indicate the enhancement of current level and the degree of reduction of denaturation. Electrochemical characterization will be performed using the ultrahigh sensitivity of a potentiostat that has current resolution on the femtoampere level. More precise characterization of electron transfer across regions of the interface will be made using in-situ scanning tunneling spectroscopy. This highly localized tunneling will be used to probe the interaction between electrons and the active centers of protein/enzyme molecules, allowing a fundamental study of electron transfer rate and hence the limiting factor for electron transfer. This project investigates an essential requirement for achieving ultrasensifivity in amperometric biosensing. Amperometric biosensors with significantly improved sensitivity are demanded in clinical and environmental applications, including detecting extremely small amounts of adrenaline in treatment of tumors, monitoring pesticides, and the realization of in-vivo and implantable subcutaneous sensors. Also, nanoscale sensing schemes provides additional improvement of sensitivity and allows measurement of small amount of samples. It is expected that the results of this project will be used as a general approach for obtaining enhanced electron transfer rate via immobilized proteins.